CN106701610B - A kind of Paenibacillus polymyxa and its cultural method and application - Google Patents
A kind of Paenibacillus polymyxa and its cultural method and application Download PDFInfo
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Abstract
The invention discloses a kind of Paenibacillus polymyxa (Paenibacillus polymyxa) and its cultural method and applications.The Paenibacillus polymyxa deposit number is CGMCC No.10062, and culture title is BD3736.Paenibacillus polymyxa of the present invention can generate a kind of antibacterial substance with wide spectrum, high fungistatic effect, significantly inhibit to a variety of gram-positive bacterias and Gram-negative bacteria.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to a kind of Paenibacillus polymyxa (Paenibacillus
) and its cultural method and application polymyxa.
Background technique
Many microorganisms of bacillus genus have prophylaxis effect and growth-promoting functions to plant, therefore in agricultural production
With good application potential.Series bacillus can produce various bioactivators, as enzyme, antibacterial material, plant hormone with
And flocculant etc., these active materials are mostly protein, polysaccharide, polypeptide etc..
Paenibacillus polymyxa since it has plant disease-proof and Plant growth promotion, to safety of human and livestock and it is pollution-free the advantages that
And be taken seriously, Environmental Protection Agency (EPA) is classified as can one of microorganism for commercial applications.Paenibacillus polymyxa generation
It thanks in product containing there are many available bioactive substances.These active materials are divided into polysaccharide, polypeptide and albumen by its structure
Matter etc., in addition there are glycoprotein, nucleoside analog, Pyrazine, hydroxy-aldehydic acids etc..By its function division, mainly antibiotic, enzyme
In addition class, plant hormone and flocculant etc. also generate the substance with functions such as anti-oxidant, stimulation are immune.
Summary of the invention
The technical problem to be solved by the present invention is to can generate Efficient antibacterial substance to overcome to lack in the prior art
Paenibacillus polymyxa this problem, provide a kind of Paenibacillus polymyxa (Paenibacillus polymyxa) and its
Cultural method and application.Paenibacillus polymyxa of the present invention can generate the antibacterial substance with wide spectrum, high fungistatic effect, and
To soda acid and temperature-insensitive, and under alkaline condition, bacteriostatic activity is greater than acidity, to a variety of gram-positive bacterias and gram
Negative bacterium significantly inhibits.
One of technical solution of the present invention: a kind of Paenibacillus polymyxa, deposit number are CGMCC No.10062.
In the present invention, the Paenibacillus polymyxa is deposited in Chinese microorganism strain preservation on November 26th, 2014
Administration committee's common micro-organisms center (CGMCC), preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode:
100101, deposit number are as follows: CGMCC No.10062, culture title are BD3736, and classification naming is Paenibacillus
polymyxa。
The two of technical solution of the present invention: a method of culture Paenibacillus polymyxa CGMCC No.10062 comprising
Following step: Paenibacillus polymyxa CGMCC No.10062 is inoculated in culture medium and is cultivated.
It is described to cultivate the training method that can be various microorganisms, including Liquid Culture, solid culture, half in the present invention
Solid culture etc., can be shaken cultivation, be also possible to fermentor submerged fermentation, preferably shaken cultivation.The oscillation training
Feeding revolving speed can be this field routine revolving speed, preferably 180 revs/min.
In the present invention, the temperature of the culture can be the temperature of this field routine, preferably 25-35 DEG C, more preferably
It is 30 DEG C.
In the present invention, time of the culture can be the duration of this field routine, preferably 1-7 days, more preferably for
4-6 days, be most preferably 5 days.
In the present invention, the culture medium can cultivate the conventional culture medium of series bacillus, including liquid for this field
Culture medium and solid medium, preferably selected from one of PDA culture medium, TYC culture medium, LB culture medium and cow's milk, more
It goodly is LB culture medium.The cow's milk can be the cow's milk of this field routine, preferably raw milk and/or skimmed milk,
It is more preferably skimmed milk.The solid content of the cow's milk can be this field routine content, preferably 2-15%, more
It is goodly 10-15%, is most preferably 10%, the percentage is that the quality of the solid content accounts for the mass body of the cow's milk volume
Product percentage.
The three of technical solution of the present invention: Paenibacillus polymyxa CGMCC No.10062 is preparing answering in antibacterial substance
With.
In the present invention, the antibacterial substance can inhibit Gram-negative bacteria and gram-positive bacteria.The gram-negative
Property bacterium is preferably comprised Escherichia coli (Escherichia coli), shigella flexneri (Shigella flexneri) and intestines
Scorching salmonella (Salmonella enteritidis)；The gram-positive bacteria is preferably comprised Listeria monocytogenes
(Listeria monocytogenes), gamboge micrococcus (Micrococcus luteus) and bacillus subtilis
(Bacillus subtilis)。
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that: the present invention provides a kind of Paenibacillus polymyxa (Paenibacillus
) and its cultural method and application polymyxa.By cultivating Paenibacillus polymyxa of the present invention, can obtain a kind of with wide
The antibacterial substance of spectrum, high fungistatic effect, significantly inhibits a variety of gram-positive bacterias and Gram-negative bacteria.
Biomaterial preservation information
Paenibacillus polymyxa BD3736 of the invention has been deposited in Chinese microorganism strain guarantor on November 26th, 2014
It hides administration committee's common micro-organisms center (CGMCC), preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode:
100101, deposit number are as follows: CGMCC No.10062, culture title are BD3736, and classification naming is Paenibacillus
polymyxa。
Detailed description of the invention
Fig. 1 is that the present invention states antibacterial series bacillus fermentation broth extract to the inhibiting effect of indicator bacteria.
Fig. 2 be antibacterial series bacillus fermentation broth extract of the present invention through Sephadex G-15 column chromatography after not
With UV absorption result of the collecting pipe at 280nm.
Fig. 3 be antibacterial series bacillus fermentation broth extract of the present invention through Sephadex G-15 column chromatography after not
With collecting pipe eluent to the bacteriostatic activity of salmonella.
Fig. 4 be antibacterial series bacillus fermentation broth extract of the present invention through Sephadex G-15 column chromatography after not
With collecting pipe eluent protein content.
Fig. 5 be antibacterial series bacillus fermentation broth extract of the present invention through Sephadex G-15 column chromatography after not
With collecting pipe eluent after ammonium sulfate precipitation, SDS-PAGE electrophoresis result.Wherein, M: albumen Marker；23-26 is represented
The collecting pipe number of Sephedx G-15 column chromatography.
Fig. 6 be antibacterial series bacillus fermentation broth extract of the present invention after SDS-PAGE electrophoresis each band to intestines
The fungistatic effect of scorching salmonella.
Fig. 7 is stability of the antibacterial series bacillus fermentation broth extract of the present invention to treatment of different temperature.
Fig. 8 is the stability of antibacterial series bacillus fermentation broth extract different pH values processing of the present invention.Figure
Middle ordinate is R2-r2(R is that inhibition zone radius caused by different pH buffers is added in sample, and r is produced by different pH buffers
Raw inhibition zone radius).
Fig. 9 is stability of the antibacterial series bacillus fermentation broth extract of the present invention to different enzymatic treatments.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification
Medium component used in the present invention is as follows:
TYC culture medium: tryptone (or casein hydrolysate) 1.5g；Yeast extract 0.5g；L-cysteine 0.02g；Sodium acetate 2.0g；
Na2SO40.01g；NaCl 0.1g；Sucrose 5.0g；Distilled water 100m L；115 DEG C, sterilize 15min.
TYC solid medium: 1.8g agar is separately added in above-mentioned TYC culture medium.
LB culture medium: tryptone (or casein hydrolysate) 1g；Yeast extract 0.5g；NaCl 0.5g, water 100mL, is sterilized by 115 DEG C
15min。
LB solid medium: 1.8g agar is added in above-mentioned LB culture medium.
PDA culture medium: potato leaching powder, 0.5g；Glucose, 2.0g；Chloramphenicol, 0.1g；Distilled water 100m L, 115 DEG C,
Sterilize 15min.
PDA solid medium: 1.8g agar is added in above-mentioned PDA culture medium.
BHI culture medium: tryptone (or casein hydrolysate) 1g；Yeast extract 0.5g；NaCl 0.5g, water 100mL, is killed by 115 DEG C
Bacterium 15min.
Sugared fermentation broth basal medium is bought from Beijing Luqiao Technology Co., Ltd..
Used strain in the present invention:
Escherichia coli (Escherichia coli), shigella flexneri (Shigella flexneri), Salmonella
Bacterium (Salmonella enteritidis), Listeria monocytogenes (Listeria monocytogenes), gamboge micrococcus
(Micrococcus luteus) and bacillus subtilis (Bacillus subtilis) are purchased from Chinese microorganism strain preservation
Administration committee's common micro-organisms center (CGMCC).
Used reagent in the present invention:
Genome DNA extracting reagent kit；Purchased from Tiangeng biochemical technology Co., Ltd.KOD plus and KOD plus 10 ×
Buffer (not magnesium chloride containing) spins (Toyobo) company purchased from Japan.
Primer is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd.
PCR product is sequenced by Shanghai Sheng Gong bioengineering Co., Ltd.
Heretofore described room temperature refers to the temperature for the operation room tested, generally 25 DEG C.
In following embodiment, the solid content percentage of the various cow's milk is that the solid quality in the cow's milk accounts for
The quality percent by volume of the cow's milk volume.
The separation of 1 BD3736 bacterial strain of embodiment
It takes the farmers' in Shanghai to pickle pickles, 1g is taken with sterile manner, 10mL sterile saline is put into and smashs to pieces, with sterile life
Reason salt water is serially diluted, and dilution is spread evenly across on solid TYC culture medium by way of coating, 30 DEG C of culture 24-48
Hour.It is multiple to choose viscous nasal mucus shape, the single colonie with good stringiness, is transferred on new solid TYC culture medium, obtains respectively
To the bacterium colony of purifying, one of them is BD3736 bacterial strain.
The identification of 2 BD3736 bacterial strain of embodiment
1, Morphological Identification
By the isolated BD3736 strain inoculated of embodiment 1 to LB solid medium, the LB containing 0.001% lysozyme
On solid medium, LB solid medium and PDA solid medium containing 3%NaCl, the percentage is to account for the LB to consolidate
The mass percent of body culture medium or the PDA solid medium quality.Under atmospheric conditions in incubator 30 DEG C of culture 18h to right
Number growth period and when bacterium colony size is stablized, colony morphological observation is carried out, mainly includes oxygen utilization, the size of bacterium colony, color, transparent
Neatly whether degree, wettability, bacterium colony surface state (whether flat, protrusion, fold, recess etc.), colony edge state (, do not advise
Then, radial etc.).
The results show that the BD3736 bacterial strain is facultative aerobic, do not grown in 0.001% lysozyme or 3%NaCl,
It is in thin layer, amoeboid mobile diffusion type on LB solid medium；It is large-scale mucus shape on PDA solid medium, cream
White to light yellow, surface is smooth, and often there is kick in center.
It is blue that methylene blue, spore staining and leather are further carried out after smear to the BD3736 bacterial strain in logarithmic growth phase
Albert'stain Albert, light microscopic observation thallus and gemma form and Gram's staining characteristic.The result shows that thallus is rod-shaped, gemma expands, and is in
Ellipse, positioned at the centre of thallus, Gram-positive.
2, analysis of physio biochemical characteristics
Analysis of The Physiological And Biochemical Properties is carried out to BD3736 bacterial strain using conventional method, pilot project and its results are shown in Table 1.
The physical and chemical experiment result of 1 BD3736 bacterial strain of table
1) take the test tube of 180mm × 16mm, forced fermentation with sugars Mycoplasma Broth Base culture medium in every test tube, high pressure sterilization, respectively plus
The sugar for entering 0.22 μm of film filtering, makes its final concentration of 50mM；
2) in the Paenibacillus polymyxa BD3736 bacterial strain 2-3 ring access 30mLLB culture medium of picking LB plate culture,
It is placed at 30 DEG C, 180r/min shaken cultivation 18h.12000rpm is centrifuged 10min and collects thallus under aseptic condition.Use physiological saline
0.9% (w/v) NaCl is washed 2 times, is resuspended in 1mL sugar fermentation broth basal medium；
3) the BD3736 bacterium that 10 μ L steps 2) are resuspended in 1mL sugar fermentation broth basal medium is inoculated with into step 1) test tube
Strain, for 24 hours, 48h observes the upgrowth situation of bacterial strain and detects the sugared fermentation results of bacterial strain for 30 DEG C of cultures.It the results are shown in Table shown in 2.
2 bacterial strain sugar of table fermentation qualification result
"+" indicates that, using sugar production acid, "-" expression does not produce acid.
The results show that mode bacterium of the physiological and biochemical property of above-mentioned BD3736 bacterial strain with Paenibacillus polymyxa
Strain is consistent, it is believed that it belongs to Paenibacillus polymyxa.
3,16s rDNA sequence homology analysis
In the BD3736 bacterial strain 2-3 ring access 30mL LB culture medium cultivated on picking LB solid medium as described above, set
At 30 DEG C, 180r/min shaken cultivation 18h.5mL bacterium solution 12000rpm centrifugation 10min is taken to collect thallus.It is mentioned by genomic DNA
Take the genomic DNA of the method extraction BD3736 bacterial strain in kit specification as gene magnification template, with bacterium 16s
RDNA universal primer, 27f:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492r:5 '-GGTTACCTTGTTACGACTT-3 ' into
Row PCR reaction.Reaction system: KOD plus10 × 2.5 μ L of buffer (not magnesium chloride containing), MgSO41.5 μ L, KOD plus
0.5 μ L, 2mM dNTP, 2.5 μ L, ddH216 μ L of O, 10 μM of 1 μ L of primer 2 7f, 10 μM of 1 μ L of primer 1492r, 1 μ of template DNA
L.Response procedures are as follows: 94 DEG C of initial denaturation 5min；30 circulations (94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 2min), 72
DEG C extend 8min.Amplified production is detected with 1% agarose gel electrophoresis, recycling target stripe sequencing, sequencing result such as sequence table
Shown in SEQ ID NO.1.Gained sequence by National Center for Biotechnology Information ncbi database (http: //
Www.ncbi.nlm.nih.gov) online Blast is compared.The results show that BD3736 bacterial strain 16s rDNA sequence of the present invention and more
Viscous series bacillus (Paenibacillus polymyxa) CF05 is closest, similitude 99%, therefore thinks BD3736 bacterial strain
Belong to Paenibacillus polymyxa.
4, pheS gene homology is analyzed
The BD3736 genomic DNA extracted using in above-mentioned steps is as gene magnification template, with bacterium pheS primer,
PheS-f:5 '-atgagcgaaaccatccaaatg-3 ', pheS-r:5 '-ttattgacgcgcaaactgctt-3 ' carries out PCR
Reaction.Reaction system: KOD plus10 × 2.5 μ L of buffer (not magnesium chloride containing), MgSO41.5 μ L, KOD plus, 0.5 μ L,
2mM dNTP 2.5μL、ddH216 μ L of O, 10 μM of 1 μ L of primer pheS-f, 10 μM of 1 μ L of primer pheS-r, 1 μ L of template DNA.
Response procedures are as follows: 94 DEG C of initial denaturation 5min；30 circulations (94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 2min)；72℃
Extend 8min.Amplified production is detected with 1% agarose gel electrophoresis, and recycling target stripe send sequencing, sequencing result such as sequence table
Shown in SEQ ID NO.2.Gained sequence by National Center for Biotechnology Information ncbi database (http: //
Www.ncbi.nlm.nih.gov) online Blast is compared.The results show that the pheS gene order of above-mentioned BD3736 bacterial strain with
Paenibacillus polymyxa strain CF05 similitude highest, but similitude is only 98%, therefore the two belongs to difference
Bacterial strain.
The antibacterial material in the source BD3736 3 Paenibacillus polymyxa of embodiment (Paenibacillus polymyxa)
It extracts
1, the culture of BD3736 bacterial strain
The BD3736 bacterial strain for taking 20% glycerol to save, is inoculated on LB solid medium, in atmospheric conditions 30 DEG C of constant temperature
48h in case.Then, it is 30 DEG C, 180r/min in 10% sterilized non-fat cow's milk that one ring single bacterium of scraping, which falls within 15mL solid content,
For 24 hours, BD3736 bacterial strain seed fermentation liquid is made in constant-temperature shaking culture.
It is 10% sterilized non-fat cow's milk that 130mL solid content is packed into 500mL triangular flask, by above-mentioned BD3736 bacterial strain
Seed fermentation liquid is inoculated with according to the inoculum concentration of 4% (v/v), and 30 DEG C, 180r/min constant temperature oscillation 5 days obtain BD3736 strain fermentation
Liquid.
2, the preparation of BD3736 strain fermentation liquid extract
By BD3736 bacterial strain fermentation liquor as described above at 60 DEG C water-bath 10min, be cooled to room temperature, 9000r/min, be centrifuged
25min collects supernatant.Under the action of magnetic agitation, it is slowly added to ammonium sulfate powder, reaches salt ion end saturation degree
80%, the percentage is the quality percent by volume that the quality of the salt accounts for the total volume of the supernatant and salt, heavy overnight
It forms sediment.Then 9000r/min is centrifuged 25min, collects sediment.Above-mentioned sediment is resuspended in deionized water to get BD3736
Strain fermentation liquid extract crude product.
The purifying of 4 BD3736 strain fermentation liquid extract of embodiment
(1) by BD3736 strain fermentation liquid extract crude product described in embodiment 3 using cutoff value be respectively 3500Da and
The bag filter of 1000Da, 4 DEG C are dialyzed overnight, the sample in collecting bag.Dialyzate is freeze-dried, freeze-dried powder is obtained.
(2) freeze-dried powder that step (1) obtains is dissolved into mass concentration 200mg/mL, applied sample amount 10mL, mistake with ultrapure water
The separation of Sephadex G-15 (column volume about 800mL) gel filtration chromatography.Eluent is the ultrapure water of pH 7.0, flow velocity 3mL/
min.Every 4min collects a pipe, every pipe about 12mL.It is detected at 280nm with albumen Ultraviolet Detector.Experimental result is shown in Fig. 2.
The above-mentioned sample being collected into is evaporated water phase.The antibacterial circle diameter to salmonella is measured, experimental result is shown in Fig. 3.
The results show that two sections of collecting pipes (being indicated respectively with A, B) of 14-16,23-30 show apparent bacteriostatic activity, and two sections of collections
Relatively, A 64min, B 96min illustrates their molecular weight relatively to the elution time of pipe.
(3) protein quantification kit is utilized, the different collecting pipes that step (2) is obtained detect protein content respectively.As a result
See Fig. 4.
The analysis of the relative molecular mass of 5 BD3736 strain fermentation liquid extract of embodiment
(1) SDS-PAGE electrophoresis.
The sample point sample collected in the collecting pipe that number is No. 23-26 in Example 4 runs SDS-PAGE glue, experimental result
See Fig. 5.The results show that there are two apparent colour bands after electrophoresis in sample, molecular weight distinguish 37kD~25kD and 10kDa with
Under.
(2) two bands for obtaining step (1), after being embathed repeatedly with sterile distilled water, test different separated bands
Bacteriostasis.Experimental result is shown in Fig. 6, the results show that having bacteriostatic activity lower than the band that the region 10kD occurs.
The stability to temperature of 6 BD3736 strain fermentation liquid extract of embodiment
By the water in 60 DEG C, 80 DEG C and 100 DEG C respectively of BD3736 strain fermentation liquid extract crude product obtained by embodiment 3
15min is handled at a temperature of 30min and 121 DEG C of bath, not thermally treated sample CK is as control.Utilize system as described in Example 2
Indicator bacteria (Bacterium enteritidis) plate obtained, adds the 100 above-mentioned samples of μ L to survey its antibacterial circle diameter respectively in Oxford cup.Experiment knot
Fruit sees Fig. 7, the results show that BD3736 bacterial strain produce antibacterial series bacillus fermentation broth extract crude product 60 DEG C, 80 DEG C,
After handling 30min at a temperature of 100 DEG C, there is no significant changes for bacteriostatic activity；Even after 121 DEG C of processing 15min, activity
Also it does not lose.It can be seen that the antibacterial series bacillus fermentation broth extract of BD3736 bacterial strain has preferable thermal stability.
Stability of the 7 BD3736 strain fermentation liquid extract of embodiment to pH value
With the buffer of different pH ranges by the BD3736 strain fermentation liquid extract crude product pH as obtained by embodiment 3 points
It is not adjusted to 3.0,4.0,5.0,6.0,7.0,8.0 and 9.0, is placed at room temperature for 2h, to correspond to the buffer of pH value for control, using such as
Indicator bacteria (Bacterium enteritidis) plate obtained described in embodiment 2 adds the 100 above-mentioned samples of μ L to survey its antibacterial respectively in Oxford cup
Loop diameter.
Experimental result is shown in Fig. 8, the results show that BD3736 strain fermentation liquid extract of the present invention is resistant to wider range
PH value, and under alkaline condition bacteriostatic activity be greater than acidity.
The stability of the enzyme of 8 BD3736 strain fermentation liquid extract of embodiment
(pH is respectively 1.5,7.5,7.5 to pepsin, trypsase, Proteinase K, the lipase of preparation 5mg/mL respectively
With 6.5), respectively take 100 μ L in centrifuge tube, be added embodiment 3 obtained by 400 μ of BD3736 strain fermentation liquid extract crude product
L makes the final concentration of 1mg/mL of enzyme.(pepsin, trypsase, Proteinase K and fat under the optimum temperature of each protease
Enzyme is respectively 40 DEG C, 37 DEG C, 55 DEG C and 26 DEG C) water bath with thermostatic control enzymatic hydrolysis 4h, setting 100 DEG C of boiling water water-bath 5min inactivates enzyme.Again will
PH value recalls to 6.0, is mended volume to same volume with 20mmol/mL PBS buffer solution, adds 400 μ L bacteriums with 100 μ L ultrapure waters
Element is control.Using indicator bacteria (Bacterium enteritidis) plate obtained as described in Example 2, add 100 μ L respectively in Oxford cup
Above-mentioned sample surveys its antibacterial circle diameter.Experimental result is shown in Fig. 9, the results show that BD3736 bacterial strain fermentation liquor of the present invention extracts
Object is sensitive to pepsin, Proteinase K part, insensitive to lipase, trypsase.Speculate that the BD3736 bacterial strain is sent out according to this
Zymotic fluid extract is protein matter.
The antimicrobial spectrum of 9 BD3736 bacterial strain fermentation liquor of embodiment
1, the preparation of indicator bacteria plate
The pathogen that 20% glycerol is saved, is transferred to BHI solid medium, 30 DEG C of culture 48h respectively.Then one is scraped
Ring single colonie is transferred in liquid B HI, and 30 DEG C of cultures are for 24 hours.12000rpm is centrifuged 5min and collects thallus, is diluted with sterile water, makes
At bacteria suspension.It is counted using blood counting chamber, it is 10 that it is dense, which to control whole beginning bacterium,6cfu/mL。
55 DEG C are cooled to BHI solid medium, mixes inverted plate, Mei Gepei with the indicator bacteria bacteria suspension prepared
It supports in ware and pours into 20mL or so culture medium.Up to indicator bacteria plate after solidifying and sufficiently drying.
2, the antimicrobial spectrum of antibacterial substance produced by BD3736 strain fermentation
With Escherichia coli (Escherichia coli), Bacterium enteritidis (Salmonella enteritidis), good fortune
Family name Shigella (Shigella flexneri) is Gram-negative bacteria representative, with bacillus subtilis (Bacillus
Subtilis), single to increase listeria spp (Listeria monocytogenes), staphylococcus aureus
(Staphylococcus aureus), gamboge micrococcus (Micrococcus luteus) are gram-positive bacteria representative.Referring to
Show and places Oxford cup on bacterium plate, then plus the BD3736 strain fermentation liquid extract crude product obtained of 100 μ L embodiment 3, inspection
Whether survey has inhibiting effect to it.
Experimental result is shown in Fig. 1, the results show that BD3736 strain fermentation liquid extract of the present invention is to Escherichia coli, will
Hayes bacterium, Bacterium enteritidis, Listeria monocytogenes, gamboge micrococcus and bacillus subtilis have obvious inhibition to make
With, and there is no inhibiting effect to staphylococcus aureus.Therefore BD3736 strain fermentation liquid extract has bacteriostatic activity, and has
There are wide spectrum, Efficient antibacterial effect.
The optimization of the fermentation time of 10 BD3736 strain fermentation liquid extract of embodiment
By the hair for the 10% sterilized non-fat cow's milk of 130ml solid content being fitted into 500ml triangular flask as described in Example 3
Zymotic fluid is continuously fermented, primary every sampling in 1 day.
It is spaced apart sterile Oxford cup on the indicator bacteria plate described in embodiment 9 etc., the above-mentioned fermentation liquid of 100 μ L is taken to be added
In Oxford cup, 2 repetitions of each sample will add excellent culture dish, after 30 DEG C of constant temperature incubation 36h, measure the diameter of inhibition zone
(mm).The results are shown in Table 3.
The fermented supernatant fluid of 3 BD3736 bacterial strain different time of table is to salmonella, Escherichia coli, shigella flexneri
Fungistatic effect
Note: inhibition zone test result judgement standard: antibacterial circle diameter (mm) > 20: extremely sensitive；Antibacterial circle diameter (mm) In
15~20: high sensitive；Antibacterial circle diameter (mm) is 10~14: middle sensitivity；Antibacterial circle diameter (mm) < 10: muting sensitive sense；Inhibition zone
Diameter (mm) is 0: insensitive.
As known from Table 3, the 5th day fermented supernatant fluid bacteriostatic activity of BD3736 bacterial strain is obviously most strong, average diameter of inhibition zone
About 13.08mm, and the antibacterial series bacillus fermentation broth extract is most strong to Bacterium enteritidis bacteriostasis, therefore this
Invention is represented using Bacterium enteritidis as indicator bacteria.
The antibacterial material in the source BD3736 11 Paenibacillus polymyxa of embodiment (Paenibacillus polymyxa)
It extracts
1, the culture of BD3736 bacterial strain
The BD3736 bacterial strain for taking 20% glycerol to save, is inoculated on LB solid medium, in atmospheric conditions 30 DEG C of constant temperature
48h in case.Then, it is 30 DEG C, 180r/min in 10% sterilized non-fat cow's milk that one ring single bacterium of scraping, which falls within 15mL solid content,
For 24 hours, BD3736 bacterial strain seed fermentation liquid is made in constant-temperature shaking culture.
In 500mL triangular flask be packed into 130mL sterilize Fresh Milk, by above-mentioned BD3736 bacterial strain seed fermentation liquid according to
The inoculum concentration of 4% (v/v) is inoculated with 25 DEG C, 180r/min constant temperature oscillation 5 days, obtains BD3736 bacterial strain fermentation liquor.The sterilizing is fresh
The solid content of cow's milk is 11%.
2, the preparation of BD3736 strain fermentation liquid extract
By BD3736 bacterial strain fermentation liquor as described above at 50 DEG C water-bath 15min, be cooled to room temperature, 9000r/min, be centrifuged
25min collects supernatant.Under the action of magnetic agitation, it is slowly added to ammonium sulfate powder, reaches salt ion end saturation degree
80%, the percentage is the quality percent by volume that the quality of the salt accounts for the total volume of the supernatant and salt.It is heavy overnight
It forms sediment.Then 9000r/min is centrifuged 25min, collects sediment.Above-mentioned sediment is resuspended in deionized water to get BD3736
Strain fermentation liquid extract crude product.
Measurement antibacterial series bacillus fermentation broth extract as described above is to the antibacterial of indicator bacteria plate (salmonella)
Loop diameter.The results show that antibacterial circle diameter is 14.5mm.
The antibacterial material in the source BD3736 12 Paenibacillus polymyxa of embodiment (Paenibacillus polymyxa)
It extracts
1, the culture of BD3736 bacterial strain
The BD3736 bacterial strain for taking 20% glycerol to save, is inoculated on LB solid medium, in atmospheric conditions 30 DEG C of constant temperature
48h in case.Then, one ring single bacterium of scraping falls within 10% sterilized non-fat cow's milk of 15mL solid content, 30 DEG C, 180r/min constant temperature
For 24 hours, BD3736 bacterial strain seed fermentation liquid is made in shaken cultivation.
In 500mL triangular flask be packed into 130mL sterilized non-fat cow's milk, by above-mentioned BD3736 bacterial strain seed fermentation liquid according to
The inoculum concentration of 4% (v/v) is inoculated with 25 DEG C, 180r/min constant temperature oscillation 5 days, obtains BD3736 bacterial strain fermentation liquor.The sterilized non-fat
The solid content of cow's milk is 2%.
2, the preparation of BD3736 strain fermentation liquid extract
By BD3736 bacterial strain fermentation liquor as described above at 50 DEG C water-bath 15min, be cooled to room temperature, 9000r/min, be centrifuged
25min collects supernatant.Under the action of magnetic agitation, it is slowly added to ammonium sulfate powder, reaches salt ion end saturation degree
80%, the percentage is the quality percent by volume that the quality of the salt accounts for the total volume of the supernatant and salt.It is heavy overnight
It forms sediment.Then 9000r/min is centrifuged 25min, collects sediment.Above-mentioned sediment is resuspended in deionized water to get BD3736
Strain fermentation liquid extract crude product.
Measurement antibacterial series bacillus fermentation broth extract as described above is to the antibacterial of indicator bacteria plate (salmonella)
Loop diameter.The results show that antibacterial circle diameter is 13mm.
The antibacterial material in the source BD3736 13 Paenibacillus polymyxa of embodiment (Paenibacillus polymyxa)
It extracts
1, the culture of BD3736 bacterial strain
The BD3736 bacterial strain for taking 20% glycerol to save, is inoculated on LB solid medium, in atmospheric conditions 30 DEG C of constant temperature
48h in case.Then, it is 30 DEG C, 180r/min in 10% sterilized non-fat cow's milk that one ring single bacterium of scraping, which falls within 15mL solid content,
For 24 hours, BD3736 bacterial strain seed fermentation liquid is made in constant-temperature shaking culture.
In 500mL triangular flask be packed into 130mL sterilized non-fat cow's milk, by above-mentioned BD3736 bacterial strain seed fermentation liquid according to
The inoculum concentration of 4% (v/v) is inoculated with 35 DEG C, 180r/min constant temperature oscillation 5 days, obtains BD3736 bacterial strain fermentation liquor.The sterilized non-fat
The solid content of cow's milk is 15%.
2, the preparation of BD3736 strain fermentation liquid extract
By BD3736 bacterial strain fermentation liquor as described above at 80 DEG C water-bath 5min, be cooled to room temperature, 9000r/min, be centrifuged
25min collects supernatant.Under the action of magnetic agitation, it is slowly added to ammonium sulfate powder, reaches salt ion end saturation degree
80%, the percentage is the quality percent by volume that the quality of the salt accounts for the total volume of the supernatant and salt.It is heavy overnight
It forms sediment.Then 9000r/min is centrifuged 25min, collects sediment.Above-mentioned sediment is resuspended in deionized water to get BD3736
Strain fermentation liquid extract crude product.
Measurement antibacterial series bacillus fermentation broth extract as described above is to the antibacterial of indicator bacteria plate (salmonella)
Loop diameter.The results show that antibacterial circle diameter is 14mm.
It should be understood that those skilled in the art can be to correlation of the invention after having read above content of the invention
Condition makes various changes or modifications, and these equivalent forms also fall within the scope of the appended claims of the present application.
Claims (10)
1. a kind of Paenibacillus polymyxa (Paenibacillus polymyxa), which is characterized in that its deposit number is CGMCC
No.10062。
2. a kind of method for cultivating Paenibacillus polymyxa as described in claim 1, which is characterized in that it includes the following steps:
Paenibacillus polymyxa CGMCC No.10062 is inoculated in culture medium and is cultivated.
3. method as claimed in claim 2, which is characterized in that the temperature of the culture is 25-35 DEG C.
4. method as claimed in claim 2, which is characterized in that the culture is shaken cultivation.
5. method as claimed in claim 4, which is characterized in that the revolving speed of the shaken cultivation is 180 revs/min.
6. method as claimed in claim 2, which is characterized in that the time of the culture is 1-7 days.
7. method as claimed in claim 2, which is characterized in that the culture medium is selected from PDA culture medium, TYC culture medium, LB training
Support one of base and cow's milk.
8. method as claimed in claim 7, which is characterized in that the cow's milk is raw milk and/or skimmed milk；And/or institute
The solid content for stating cow's milk is 2-15%, and the percentage is that the quality of the solid content accounts for the mass body of the cow's milk volume
Product percentage.
9. Paenibacillus polymyxa as described in claim 1 is preparing the application in antibacterial substance.
10. application as claimed in claim 9, which is characterized in that the antibacterial substance inhibit Escherichia coli (Escherichia coli), Bacterium enteritidis (Salmonella enteritidis), shigella flexneri (Shigella flexneri), it is withered
Careless bacillus (Bacillus subtilis), it is single increase listeria spp (Listeria monocytogenes) and gamboge it is small
Coccus (Micrococcus luteus)。
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| CN107788119A (en) * | 2017-10-31 | 2018-03-13 | 光明乳业股份有限公司 | A kind of application for the method and the Paenibacillus polymyxa that acidified milk is prepared using Paenibacillus polymyxa |
| CN107760724A (en) * | 2017-10-31 | 2018-03-06 | 光明乳业股份有限公司 | A kind of method and the α glucosidase inhibitors that α glucosidase inhibitors are prepared using Paenibacillus polymyxa |
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| CN110055188A (en) * | 2019-03-07 | 2019-07-26 | 南京师范大学 | One plant of Paenibacillus polymyxa XW4 for producing bacteriostatic peptide and its separation screening and application |
| CN113913345A (en) * | 2021-11-18 | 2022-01-11 | 河南省农业科学院小麦研究所 | Microbial agent for promoting yield increase and quality improvement of cereal crops and application thereof |
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